Herpes simplex virus (HSV) infection of many cultured cells, such as Vero cells, is initiated by receptor-binding and fusion with the cell surface. A survey of HSV entry into diverse cell lines indicated that Chinese hamster ovary (CHO) and HeLa cells support an endocytic, pH-dependent entry pathway for HSV. In these cell types, but not Vero cells, lysosomotropic agents, such as the weak base ammonium chloride and the ionophore monensin which buffer the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early, postbinding step during infection by several strains of both serotypes. CHO cell entry mediated by the HSV cellular receptors HveA, nectin-1 or nectin-2 was blocked by these agents. Enveloped virions were readily detected in vesicles by electron microscopy (EM) at < 30 min post-infection. Depletion of cellular energy, which blocks endocytosis, prevented cellular uptake and infection by HSV in CHO and HeLa cells. Entry into Vero cells was unaffected by energy depletion, and as expected, EM images of virus fusion with the Vero cell surface were prevalent. The kinetics of HSV entry into all three cell lines was similar, which suggests that entry is equally efficient regardless of the pathway utilized. The pH-dependent fusion function of many viruses is inactivated by acid pre-treatment of virions. Entry of HSV into all cell types was reduced by 50% following treatment with pH 5.5. This inactivation was rapid (< 1 min), temperature-dependent, and irreversible. Acid pretreatment had no detectable effect on the antigenic conformation of gD, the receptor-binding glycoprotein, and no effect on binding of radiolabelled virions to cellular receptors. This suggests that mildly acidic pH inactivates a post-binding event in entry such as membrane fusion. We propose that HSV has the capacity to efficiently enter cells by endocytic uptake followed by a low pH-triggered step.